Human induced-T-to-natural killer cells have potent anti-tumour activities

Zhiwu Jiang, Le Qin, Yuou Tang, Rui Liao, Jingxuan Shi, Bingjia He, Shanglin Li, Diwei Zheng, Yuanbin Cui, Qiting Wu, Youguo Long, Yao Yao, Zhihui Wei, Qilan Hong, Yi Wu, Yuanbang Mai, Shixue Gou, Xiaoping Li, Robert Weinkove, Sam Norton, Wei Luo, Weineng Feng, Hongsheng Zhou, Qifa Liu, Jiekai Chen, Liangxue Lai, Xinwen Chen, Duanqing Pei, Thomas Graf, Xingguo Liu, Yangqiu Li, Pentao Liu, Zhenfeng Zhang, Peng Li, Zhiwu Jiang, Le Qin, Yuou Tang, Rui Liao, Jingxuan Shi, Bingjia He, Shanglin Li, Diwei Zheng, Yuanbin Cui, Qiting Wu, Youguo Long, Yao Yao, Zhihui Wei, Qilan Hong, Yi Wu, Yuanbang Mai, Shixue Gou, Xiaoping Li, Robert Weinkove, Sam Norton, Wei Luo, Weineng Feng, Hongsheng Zhou, Qifa Liu, Jiekai Chen, Liangxue Lai, Xinwen Chen, Duanqing Pei, Thomas Graf, Xingguo Liu, Yangqiu Li, Pentao Liu, Zhenfeng Zhang, Peng Li

Abstract

Background: Adoptive cell therapy (ACT) is a particularly promising area of cancer immunotherapy, engineered T and NK cells that express chimeric antigen receptors (CAR) are being explored for treating hematopoietic malignancies but exhibit limited clinical benefits for solid tumour patients, successful cellular immunotherapy of solid tumors demands new strategies.

Methods: Inactivation of BCL11B were performed by CRISPR/Cas9 in human T cells. Immunophenotypic and transcriptional profiles of sgBCL11B T cells were characterized by cytometer and transcriptomics, respectively. sgBCL11B T cells are further engineered with chimeric antigen receptor. Anti-tumor activity of ITNK or CAR-ITNK cells were evaluated in preclinical and clinical studies.

Results: We report that inactivation of BCL11B in human CD8+ and CD4+ T cells induced their reprogramming into induced T-to-natural killer cells (ITNKs). ITNKs contained a diverse TCR repertoire; downregulated T cell-associated genes such as TCF7 and LEF1; and expressed high levels of NK cell lineage-associated genes. ITNKs and chimeric antigen receptor (CAR)-transduced ITNKs selectively lysed a variety of cancer cells in culture and suppressed the growth of solid tumors in xenograft models. In a preliminary clinical study, autologous administration of ITNKs in patients with advanced solid tumors was well tolerated, and tumor stabilization was seen in six out nine patients, with one partial remission.

Conclusions: The novel ITNKs thus may be a promising novel cell source for cancer immunotherapy.

Trial registration: ClinicalTrials.gov, NCT03882840 . Registered 20 March 2019-Retrospectively registered.

Keywords: BCL11B; CRISPR/Cas9; Immunotherapy; T cells.

Conflict of interest statement

P. L. is a scientific founder of GZI and has equity in GZI.

© 2022. The Author(s).

Figures

Fig. 1
Fig. 1
Reprogramming of primary human T cells into ITNKs by inactivating BCL11B. A sgRNA targeting exon 2 and exon 3 of the BCL11B locus. sgRNA, Cas9 and EGFP elements were integrated into a single vector. B Western blot analysis of BCL11B (120 kDa) levels in three representative samples of CB-derived T cells that were transduced with sgCtrl or NKp46+CD3+ cells (purity: 92.41 ± 2.60%) that were sorted from sgBCL11B-engineered T cells. C Representative flow cytometric detection of CD3, CD56, NKp30 and NKp46 in T cells: T cells transduced with sgCtrl, T cells transduced with sgBCL11B and normal NK cells (CD3−CD56+). Data are representative of five independent experiments. D Graph summarizing the percentages of CD56+, NKp30+, and NKp46+ cells in CD3+ T cells that received sgBCL11B or sgCtrl at 14 days post electroporation. The mean values of five independent healthy donors are shown. P < 0.001 for CD56+, NKp30+ and NKp46+ T cells in sgBCL11B-electroporated T cells compared to sgCtrl-electroporated T cells. ***P ≤ 0.001, two-way ANOVA with Sidak’s multiple comparisons test. E TCR diversity in sgCtrl T and NKp46+CD3+ cells purified from sgBCL11B-edited T cells from the same donor based on variable chain sequencing data for the TCRβ locus. The 20 variable chain sequences at the TCRβ locus were analyzed
Fig. 2
Fig. 2
Immunophenotypic characteristics of ITNK cells. A Representative CyTOF analysis of the immunophenotypic profiles of CB-derived T cells on Day 2 to Day 10 post electroporation with sgBCL11B. B, C UMAP plots with colored circles highlighting the cells at different time points (Day 2, Day 4, Day 6, Day 8, and Day 10) (B) and different clusters of T cells based on their immunophenotypic profiles (C). D Representative T cell marker (CD4, CD8A, CD62L, CCR7, and γδTCR) and NK cell-associated marker (CD56, NKp30, NKp44, NKp46, CD11c and CD16) expression in various subtypes of T cells and ITNKs. E Representative flow cytometric detection of CCR1, CCR3, CCR6, CCR8, and CXCR4 in T cells (CD3+CD4+/CD8+), ITNKs (CD3+CD4+NKp30+/CD3+CD8+NKp46+) and normal NK cells (CD3−CD56+). Data are representative of three independent experiments. F Graph summarizing the percentages of CCR1, CCR3, CCR6, CCR8, and CXCR4 cells in T cells, ITNKs and NK cells after 14 days of culture. The results represent the mean ± SD. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001; one-way ANOVA with Tukey’s multiple comparisons test
Fig. 3
Fig. 3
ITNKs acquire transcriptional profiles of NK cells. A UMAP visualization of sgBCL11B-transduced T cells after batch correction, colored by samples collected for scRNA-seq analysis on Day 2 (D2), Day 4 (D4), Day 6 (D6), Day 8 (D8), and Day 10 (D10) post electroporation. B UMAP visualization of sgBCL11B-transduced T cells mainly classified into 6 different clusters based on their gene expression profiles. C Plots showing the expression levels of selected genes associated with T (CD3E, CD8A and CD4) or NK cell lineages (NCAM1, NCR3, ID2, IL2RB, and NFIL3) in sgBCL11B-transduced T cells based on scRNA-seq analysis. D Violin plots showing the expression levels of NK cell- and T cell-associated genes, AP-1 family genes, glycolysis-associated genes and genes regulating proliferation in activated T cells (Cluster 0 (CD4 T) and Cluster 3 (CD8 T)), effector T cells (Cluster 1 (CD4 T) and Cluster 4 (CD8 T)), and ITNKs (Cluster 2 (CD4 ITNK) and Cluster 5 (CD8 ITNK)). E T cells and ITNKs were purified (purity > 90%) from sgCtrl- and sgBCL11B-electroporated T cells from Donors 1 and 2. Purified NK cells (CD3−CD56+) (purity > 90%) were enriched from NK cell cultures from Donors 1–3. Cells from Donors 1–3 were collected from CB samples. Principal component analysis (PCA) was used to evaluate the similarities in global gene expression profiles among purified ITNKs, NK cells, and T cells. F Differential expression patterns of selected NK cell-, glycolysis-, and T cell-associated genes and AP-1 family genes differentially expressed in ITNKs, NK cells, and T cells based on RNA-seq analysis
Fig. 4
Fig. 4
Evaluating the antitumor effects of ITNKs in vitro and in vivo. A The bar chart shows IFN-γ secretion by T cells, ITNKs and NK cells after activation for 24 h with the indicated antibodies (anti-NKp30, anti-NKp46 and anti-CD3/CD28 at 5 μg/ml). Samples were collected from three individual donors. Data are shown as the mean ± SD; *P ≤ 0.05 and ***P ≤ 0.001; two-way ANOVA with Tukey’s multiple comparisons test. B Cytokine secretion profiles of ITNKs, T cells, and NK cells in coculture with K562 cells. ITNKs, T cells, and NK cells were incubated with K562 cells at an E:T ratio of 1:1 for 24 h. The supernatants were then harvested, and the concentrations of the indicated cytokines were measured by a multiplex immunoassay. The values shown represent the mean ± SD of 3 different donors. **P ≤ 0.01 and ***P ≤ 0.001; two-way ANOVA with Tukey’s multiple comparisons test. C Killing assays showing the percent cytotoxicity of ITNKs, NK cells and T cells against K562 cells. The data are shown as the mean ± SD; P < 0.0001 (ITNKs vs. T cells for K562 cells) and ***P ≤ 0.001; two-way ANOVA with Tukey’s multiple comparisons test. D The percent cytotoxicity of ITNKs and T cells against NALM-6 and OTK3-overexpressing NALM-6 cells in coculture. The results were obtained from three independent experiments. P < 0.0001 (ITNKs lysing OKT3+ NALM-6 cells vs. ITNKs lysing NALM-6 cells) and ***P ≤ 0.001; two-way ANOVA with Tukey’s multiple comparisons test. E Bioluminescence images showing the fate of K562 cells transplanted into NSI mice at the indicated time points. F Quantification of the total flux analyzed by in vivo bioluminescence imaging of luciferase activity (n = 5, per group). The results represent the mean ± SD; P = 0.0347 (ITNKs vs. T cells) and *P ≤ 0.05; two-way ANOVA with Tukey’s multiple comparisons test. G Survival analysis of mice treated with PBS (n = 10), T cells (n = 15), ITNKs (n = 15), or NK cells (n = 5); P = 0.0001 (ITNKs vs. T cells) by the log-rank Mantel-Cox test. H Tumor progression of patient-derived HCC xenografts (donor: 47-year-old female with grade IV hepatocellular carcinoma) treated with PBS, T cells, ITNKs, or NK cells (n = 5). Data are shown as the mean ± SD; P = 0.0001 (ITNKs vs. T cells) by two-way ANOVA with Tukey’s multiple comparisons test, ***P ≤ 0.001
Fig. 5
Fig. 5
Enhanced anti-tumour activity in ITNKs engineered with CAR. A Representative flow cytometer analysis of CAR-ITNKs (CAR19-ITNKs and CARGPC3-ITNKs) which were defined as GFP + CD3 + NKp46+ on day 14 post electroporation; (B) ITNKs and T cells expressing CAR19 were co-cultured with K562 cells that expressed CD19 with the indicated ratios of effectors to targets. The percentage means of specific tumour cell lysis ± SD are shown. P < 0.0001 (CAR19-ITNK vs. CAR19-T) and P < 0.0001 (CAR19-ITNK vs. ITNK); ***P ≤ 0.001; two-way ANOVA with Tukey’s multiple comparisons test; (C) ITNK and T cells expressing CARGPC3 were co-cultured with HepG2-GL with the indicated effector to target ratios. The means of percentages of specific tumour cell lysis ± SD are shown. P < 0.0001 (CARGPC3-ITNK vs. CARGPC3-T), ***P ≤ 0.001; two-way ANOVA with Tukey’s multiple comparisons test; (D) Tumour progression of HCC xenografts treated with T, ITNK, CARGPC3-T and CARGPC3-ITNK cells (n = 5, per groups) on day 0 and day 3 when the volumes of tumours were about 50mm3. Data are shown as mean ± SD; P < 0.0001 (ITNK vs. T) and P = 0.0015 (CARGPC3-ITNK vs CARGPC3-T); two-way ANOVA with Tukey’s multiple comparisons test, **P ≤ 0.01 ***P ≤ 0.001
Fig. 6
Fig. 6
Safety and potency of ITNK infusions in cancer patients. A Serum IL-6 levels in patients (GD001-GD009) from day 1 to 3 months post ITNK infusion. B Cytokine levels in patients on day 0 and day 3 post ITNK infusion. PDGF-BB, MIP-1β, GM-CSF, M-CSF and TNF-α were significantly elevated in patients (GD002, GD004-GD007) after ITNK infusion on day 3 based on Benjamini–Hochberg-adjusted p value. C Representative CT scans and PET-CT (MRI) of patients before (baseline) and after ITNK infusion at indicated time points post initial ITNK infusion. Tumors in patients GD004, GD005, GD006, GD007 and GD008 were stabilized (all tumors increased less than 20% in size), and GD002 achieved partial remission (PR), as revealed by both CT and PET-CT scan imaging diagnostic analysis at 16 months (34.6% decrease in tumor size), whereas tumors in patients GD001, GD003 and GD009 progressed following ITNK treatment (all tumors increased more than 30% in size). Red circles indicate the monitored tumor sites. D Percentages of ITNK cytotoxicity for patients with progressing diseases and patients with stable diseases. The assay was performed against K562 cells at an E: T ratio equal to 1:1 measured 24 h after coculture. Data are shown as the mean ± SD; P = 0.0082 (stable vs. progressing); unpaired t test, **P ≤ 0.01

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